Pro-Resolving FPR2 Agonists Regulate NADPH Oxidase-Dependent Phosphorylation of HSP27, OSR1, and MARCKS and Activation of the Respective Upstream Kinases

Abstract

Background: Formyl peptide receptor 2 (FPR2) is involved in the pathogenesis of chronic inflammatory diseases, being activated either by pro-resolving or proinflammatory ligands. FPR2-associated signal transduction pathways result in phosphorylation of several proteins and in NADPH oxidase activation. We, herein, investigated molecular mechanisms underlying phosphorylation of heat shock protein 27 (HSP27), oxidative stress responsive kinase 1 (OSR1), and myristolated alanine-rich C-kinase substrate (MARCKS) elicited by the pro-resolving FPR2 agonists WKYMVm and annexin A1 (ANXA1).

Methods: CaLu-6 cells or p22phox^Crispr/Cas9 double nickase CaLu-6 cells were incubated for 5 min with WKYMVm or ANXA1, in the presence or absence of NADPH oxidase inhibitors. Phosphorylation at specific serine residues of HSP27, OSR1, and MARCKS, as well as the respective upstream kinases activated by FPR2 stimulation was analysed.

Results: Blockade of NADPH oxidase functions prevents WKYMVm- and ANXA1-induced HSP-27(Ser82), OSR1(Ser339) and MARCKS(Ser170) phosphorylation. Moreover, NADPH oxidase inhibitors prevent WKYMVm- and ANXA1-dependent activation of p38MAPK, PI3K and PKCδ, the kinases upstream to HSP-27, OSR1 and MARCKS, respectively. The same results were obtained in p22phox^Crispr/Cas9 cells.

Conclusions: FPR2 shows an immunomodulatory role by regulating proinflammatory and anti-inflammatory activities and NADPH oxidase is a key regulator of inflammatory pathways. The activation of NADPH oxidase-dependent pro-resolving downstream signals suggests that FPR2 signalling and NADPH oxidase could represent novel targets for inflammation therapeutic intervention.

Keywords: HSP-27; MARCKS (Myristolated Alanine-Rich C-Kinase Substrate); NADPH oxidase (Nicotinamide Adenine Dinucleotide Phosphate oxidase); OSR1 (Oxidative-Stress-Responsive kinase 1); annexin A1; formyl peptide receptors; inflammation; reactive oxygen species.

Figure 1

NADPH oxidase blockade functions prevents WKYMVm-induced heat shock protein 27 (HSP27), oxidative stress responsive kinase 1 (OSR1) and myristolated alanine-rich C-kinase substrate (MARCKs) phosphorylation. (a–c) CaLu-6 cells were serum deprived for 24 h and stimulated with WKYMVm for 5 min or preincubated with 100 μM Apocynin or 5 mM N-acety-l-cysteine (NAC) before stimulation; (d–f) CaLu-6-control^Crispr/Cas9 cells (CTR) and p22phox^Crispr/Cas9 (p22phox^Crispr) cells were serum starved for 24 h, and then stimulated with a scrambled hexapeptide or with 10 μM WKYMVm for 5 min. Sixty micrograms of whole lysates were resolved on 10% SDS-PAGE, transferred onto PVDF membrane and immunoblotted with (a,d) anti-phospho-HSP27(Ser82) (α-pHSP27(S82)), or (b,e) anti-phospho-OSR1(Ser339) (α-pOSR1(S339)), or (c,f) anti-phospho-MARCKS(Ser170) (α-pMARCKS(S170)) antibodies. An antitubulin (α tubulin) antibody was used as a control for protein loading. Bar graphs show the densitometric analysis performed on phosphorylated bands. Data are representative of at least three independent experiments. * p < 0.05 as compared with unstimulated cells.

Figure 2

Annexin A1 (ANXA1)-induced HSP27, OSR1, and MARCKs phosphorylation depends on NADPH oxidase activity. (a–c) Serum-starved CaLu-6 cells were stimulated with 10 nM ANXA1 for 5 min in the presence or absence of the appropriate amounts of NADPH oxidase inhibitors; (d–f) CaLu-6-control^Crispr/Cas9 cells (CTR) and p22phox^Crispr/Cas9 (p22phox^Crispr) cells were grown until they reached 80% of confluence, serum deprived for 24 h, and then stimulated with the vehicle or with 10 nM ANXA1 for 5 min. Total lysates (60 μg) were resolved on 10% SDS-PAGE and membranes were incubated with (a,d) anti-phospho-HSP27(Ser82) (α-pHSP27(S82)), or (b,e) anti-phospho-OSR1(Ser339) (α-pOSR1(S339)), or (c,f) anti-phospho-MARCKS(Ser170) (α-pMARCKS(S170)) antibodies. An antitubulin (α-tubulin) antibody was used as a control for protein loading. The data are representative of at least three independent experiments. Densitometric analysis was performed as described in Materials and Methods. * p < 0.05 as compared with unstimulated cells.

Figure 3

WKYMVm- and ANXA1-induced p38MAPK activation depends on NADPH oxidase-induced reactive oxygen species (ROS) generation. Serum-deprived CaLu-6 cells were stimulated with (a) 10 μM WKYMVm or (b) 10 nM ANXA1 for 5 min or preincubated with 100 μM apocynin or 5 mM NAC before stimulation. The CaLu-6-control^Crispr/Cas9 cells (CTR) and p22phox^Crispr/Cas9 (p22phox^Crispr) cells were serum starved for 24 h, and then stimulated with (c) a scrambled hexapeptide or 10 μM WKYMVm for 5 min, or (d) the vehicle or 10 nM ANXA1 for 5 min. Whole lysates (60 μg) were resolved on 10% SDS-PAGE, transferred onto PVDF membrane, and incubated with an anti-phospho-p38MAPK (T180/Y182) antibody (α-p38MAPK). The data are representative of at least three independent experiments. Bar graphs show the densitometric analysis performed on phosphorylated bands. * p < 0.05 as compared with unstimulated cells. An antitubulin (α-tubulin) antibody was used as a control for protein loading.

Figure 4

PI3K phosphorylation triggered by WKYMVm or ANXA1 signalling is prevented by NADPH oxidase inhibition. CaLu-6 cells were grown until they reach 80% of confluence, and then serum deprived for 24 h. Cells were stimulated with (a) 10 μM WKYMVm or (b) 10 nM ANXA1 for 5 min, or preincubated with 100 μM apocynin or 5 mM NAC before stimulation. Serum-starved CaLu-6-control^Crispr/Cas9 cells (CTR) and p22phox^Crispr/Cas9 (p22phox^Crispr) cells were stimulated with (c) a scrambled hexapeptide or 10 μM WKYMVm for 5 min, or (d) the vehicle or 10 nM ANXA1 for 5 min. Total extracts were electrophoresized on 10% SDS-PAGE and membranes were incubated with an anti-phospho-PI3K (Y458) antibody (α-pPI3K). An antitubulin (α-tubulin) antibody was used as a control for protein loading. Bar graphs show the densitometric analysis performed on phosphorylated bands, as described in Materials and Methods. * p < 0.05 as compared with unstimulated cells. The experiments are representative of at least three independent assays.

Figure 5

WKYMVm- and ANXA1-induced PKCδ activation depends on NADPH oxidase-dependent ROS generation. Serum-starved CaLu-6 cells were stimulated with (a) 10 μM WKYMVm or (b) 10 nM ANXA1 for 5 min, in the presence or absence of the appropriate amounts of NADPH oxidase inhibitors. CaLu-6-control^Crispr/Cas9 cells (CTR) and p22phox^Crispr/Cas9 (p22phox^Crispr) cells were serum deprived for 24 h, and then stimulated with (c) a scrambled hexapeptide or 10 μM WKYMVm, or (d) the vehicle or 10 nM ANXA1 for 5 min. Sixty micrograms of whole extracts were resolved on 10% SDS-PAGE, transferred onto PVDF membrane, and incubated with an anti-phospho-PKCδ(Thr507) antibody ((α-pPKCδ(T507)). An antitubulin (α-tubulin) antibody was used as a control for protein loading. Bar graphs show the densitometric analysis performed on phosphorylated bands. Data are representative of at least three independent experiments. * p < 0.05 as compared with unstimulated cells.